BRPI0808716B1 - Methods for producing transformed cotton plant tissue - Google Patents

Abstract

MERISTEM EXCISION AND TRANSFORMATION METHOD. The present invention relates to the excision of explant material comprising cottonseed meristematic tissue. Methods for preparing, storing, transforming, and selecting or identifying tissues of transformed plants, how tissues from transformable meristems and plants are produced by such methods, and apparatus for preparing tissues are described.

Description

Background of the Invention

[001] This Patent Application claims priority to Provisional Patent Applications US Nos. 60/894,096, filed March 9, 2007, and US 60/915,066, filed April 30, 2007, the entire contents of which are here incorporated as a reference.

Technical Field of Invention

[002] The present invention relates to methods for preparing and transforming cotton meristematic plant tissue and subsequent regeneration of transgenic plants.

Description of Prior Techniques

[003] Transformed plants can be obtained by delivering heterologous DNA to meristematic tissue of a plant embryo. The meristematic tissue contains formative plant cells that differentiate to produce multiple plant structures, including stems, roots, leaves, germ-line tissue, and seeds. Plant meristems can be treated, and selected or screened to determine which of these treated meristems have incorporated new genetic information into the germ-line tissue. US Patent Nos. 6,384,301 and 7,002,058, and Patent Application Publication No. 2006/0059589 describe methods for generically transforming soybeans (Glycine max) using bacterially mediated gene transfer directly into meristematic cells of soybean embryos. . US Publication No. 2005/0005321 describes the excision of meristematic tissues from seeds. Isolated cotton meristems and shoot apex tissues were transformed (see, for example, WO No. 9215675; US Patent No. 5,164,310; McCabe and Martinell, 1993). However, due to physical and physiological properties of cotton seeds, the conditions for excision of meristematic material and transformation to obtain transgenic plants differ from those for soybean.

[004] The current manual process of excision of embryos from soaked cottonseeds is slow and has an ergonomic burden. In this process, surface-sterilized seeds are handled aseptically at one time with gloved hands. The explant is then carefully excised. In the case of cotton meristems, the seeds are carefully oriented in such a way as to eject the embryo with applied force. Even with careful handling of individual seeds, poor recovery of usable embryos is common.

[005] Bacterial contamination of embryos after excision is also a significant concern. Greater handling to preserve higher viability and explant recovery also increases the possibility of destructive contamination (which will manifest in subsequent processing steps). Such contamination can result in significant loss, as a single contaminated explant will contaminate other samples during tissue transformation and culture. This causes loss of yield and/or transformation frequency. Furthermore, the manual excision process is extremely labour-intensive, time consuming, and represents a barrier to an increased production volume of the transformation process, in which many plants must typically be treated with desired yield results. There remains, therefore, a great need for a process that would increase the availability of transformable cotton embryos without unacceptably increasing total costs and/or lead times for preparing explants for transformation.

Summary of the Invention

[006] In one aspect, the invention provides a high-volume production method for producing processed cotton fabric, comprising: (a) mechanically breaking cotton seeds to obtain a plurality of embryonic cotton meristem explants; and (b) contacting the explants with a selected DNA sequence to obtain at least a first explant transformed with the selected DNA. In certain embodiments, explants are stored at a temperature between 0-15°C for between 1 hour and 7 days before being contacted with a selected DNA sequence. In other embodiments, the method further comprises the step of regenerating a transgenic cotton plant transformed with the DNA selected from the at least first explant. In certain embodiments, the method does not comprise generating a callus culture from the explant.

[007] In specific embodiments, the transgenic cotton plant originates from the transformation of a meristem, which results in the transformation of germ-line tissue. In certain embodiments, the resulting plant is non-chimeric. In alternative embodiments, the resulting plant is chimeric. In certain embodiments, the method further comprises screening explants before contacting the explants with the selected DNA sequence to identify a fraction of explants susceptible to transformation with the selected DNA and regenerating a transgenic plant therefrom. In other embodiments, sorting the explants comprises mechanically placing the ruptured cotton seeds, which comprise the explants, into an aqueous environment, and selecting the explants to contact the selected DNA, based on flotation. In still other embodiments, sorting the explants comprises mechanically sieving the ruptured cottonseeds to remove the explants from the seed coat or cotyledon tissue. Screening explants can also enrich the fraction of explants susceptible to transformation.

[008] In certain embodiments, the selected DNA sequence encodes a selectable or screenable marker, or it may encode or otherwise specify an agronomic trait, including environmental adaptability, among other phenotypes. The trace can also specify the production of a desired end product. The method may further comprise selecting or screening for an explant transformed with the selected DNA, placing the explant in contact with a selective agent, where the selectable marker confers tolerance to the selective agent. The method can be defined as comprising regenerating transgenic plant tissue from the explants. The method may be further defined as comprising regenerating chimeric transgenic plant tissue from the explant and selecting or screening for transgenic tissue from the plant tissue. In other embodiments, the method further comprises regenerating a chimeric plant from the explant and selecting or screening for transgenic tissues from the plant. In specific embodiments, transgenic cotton plant tissue originates from meristem transformation. In certain embodiments, the transgenic cotton plant originates from periclinal transformation.

[009] The method may further refer to selecting or screening for transgenic tissue, comprising placing the tissue in contact with a selective agent or an agent that produces a phenotype capable of being screened, selected from the group consisting of glufosinate, dicamba, glyphosate, spectinomycin, streptomycin, kanamycin, G418, paromomycin, hygromycin B, imidazolinone, a substrate of GUS, and combinations thereof.

[0010] In other embodiments, the method comprises mechanically breaking the cotton seeds by passing the seeds through rollers that crush the seed. In specific embodiments, the rollers comprise secondary grooves. In still other embodiments, the rollers are made of stainless steel.

[0011] In specific embodiments, explants are placed in contact with a selected DNA sequence, for example, by placing explants in contact with a Rhizobium or Agrobacterium spp. recombinant capable of transforming at least a first cell of the explant with the selected DNA. In specific embodiments, the explant is contacted by an Agrobacterium culture grown to an OD660 between about 0.0045 and about 1.4. The pH at which the meristematic cotton fabric is contacted by Agrobacterium cells can be, in certain embodiments, between about 5.0 and about 6.0, up to about 10.0. In some embodiments, Rhizobium or Agrobacterium spp. are suspended in the presence of a selective agent active against an untransformed explant before placing the explants in contact with a Rhizobium or Agrobacterium spp. recombinant. In other embodiments, explants can be contacted with a selected DNA sequence by microprojectile bombardment.

[0012] In certain embodiments, after placing explants in contact with a selected DNA sequence, the explants are grown in the presence of a selective agent at 35°C, or are grown under lighting conditions that allow normal plastid development. . In other embodiments, explants can be grown in the dark. In specific embodiments, growth at 35°C is carried out for about 1-7 days, such as about 3-5 days; the selective agent is selected from the group consisting of spectinomycin, streptomycin, kanamycin, glyphosate, glufosinate, hygromycin, and dicamba; or explants are grown under a light intensity > 5μ Einsteins, including 5-200 μ Einsteins, 5-130 μ Einsteins, or 70-130μ Einsteins with a photoperiod of about 16 light/8 dark hours.

[0013] In some embodiments, explants are grown in the presence of a fungicide before, during, or after placing the explants in contact with a selected DNA. In certain embodiments, explants are grown in the presence of a fungicide and DMSO. In specific embodiments, explants are grown in the presence of nystatin, thiabendazole, and DMSO. In another aspect, the invention provides an apparatus for the high volume production generation of transformable plant tissue comprising spaced apart rollers which comprise secondary grooves for applying a force to cotton seeds passing through the rollers. In certain embodiments, the rollers are spaced apart from each other by between about 2 mm and about 4 mm apart, or about 2.2 mm to about 3.5 mm apart, measured by the spacing of opposing roller peaks. . In specific embodiments, the rollers are made of stainless steel. The apparatus may further comprise a separator for separating the meristematic cotton explants from the seed coat or cotyledon tissue. In a specific embodiment, the separator comprises a liquid container for determining the float of transformable plant tissue. The apparatus may further comprise a water and seed containment tray. In specific embodiments, the apparatus may further comprise an automated mechanism for cleaning the apparatus, which controls one or more of the following steps: (a) applying a sanitizing solution; (b) removing the sanitizing solution; and (c) store the device under sterile conditions.

Brief Description of Drawings

[0014] The drawings that follow form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by referring to the drawing in combination with the detailed description of the specific embodiments presented herein.

[0015] FIGURE 1: Seed excision machine: (A) Seed excision machine, overview; (B) feeder; (C) top view of the feeder and rollers; (D) close-up of steel rollers, illustrating "peak to valley" clearance distance; (E) side view of rollers.

[0016] FIGURE 2: excision and purification of cotton embryos by machine: (A) effect of the size of the distance between rollers on the excision of embryos; (B) effect of sieving and flotation purification on the purity of the explant fraction; (C) Close-up of excised cotton embryo.

[0017] FIGURE 3: cross section of the sieve (V technology) with (A) schematic side view; (B) top views; and (C) background.

[0018] FIGURE 4: excision machine with water and seed containment system.

[0019] FIGURE 5: Spectinomycin as a selection agent and visual marker for early identification of transformation. (A-B) Two views of chimeric transformed cotton fabric, illustrating that untransformed fabrics appear bleached and often malformed upon selection with spectinomycin, while transformed fabrics are green and develop properly. The surviving transformed shoots are also illustrated.

[0020] FIGURE 6: Southern blot hybridization results confirming transformation, and identification of machine-excised transgenic cotton tissue.

[0021] FIGURE 7: Western blot, two exposures, demonstrating DMO transgene expression in cotton tissue derived from a machine excised explant.

[0022] FIGURE 8: Increase in Transformation Frequency (TF) % seen from the addition of increasing concentrations of spectinomycin to the co-culture medium.

Detailed Description of the Invention

[0023] The text that follows is a detailed description of the invention provided to assist those skilled in these techniques in practicing the present invention. Those skilled in the art may make modifications and variations to the embodiments described herein without departing from the spirit or scope of the present invention.

[0024] The invention provides methods and compositions for preparing, screening and using cotton explants in automated high volume production procedures. These explants can then be transformed by a selected heterologous DNA sequence, and transgenic cotton plants can be regenerated from them, without the need to generate a callus cell culture from the transformed explant, to obtain transgenic progeny plants. The selected heterologous DNA sequence may, for example, encode a marker capable of being screened or selectable, and/or comprise a gene of interest that specifies a trait displayed by a plant or cotton cell resulting from expression of the heterologous nucleic acid. The trait can be agronomically useful, for example, resulting in higher yields, resistance to pests or pathogens, or environmental adaptability, among other phenotypes. This allows for a fast and efficient mechanized high-volume production process to generate transformed cotton plants. The mechanized process for excision of cotton described provides monetary, safety and flexibility benefits. Mechanization reduces the number of man-hours needed to produce 10,000 cotton explants from around 40 to just 2.4 hours, significantly saving labor costs. A lower cost of explants produces greater opportunities for the development of improved transformation methods or the use of techniques that, while providing benefits such as reduced time to create transgenic plants or the ability to use better cultivars for transformation, have to date produced a transformation efficiency too poor to be widely implemented. Such a technique allows for larger numbers of transgenes to be tested and higher quality episodes to be chosen for further analysis, as only a small number of transformation episodes are expected to present the most desired expression profiles, suitable for commercial development. An improved excision process also allows for better synchronization and programming of transformation steps, because of greater flexibility in explant distribution.

[0025] The mechanical process described here can be easily increased to support transformation with higher production volume. The potential production rate, assuming 28 h of excision per week, is increased from 7,000 explants per person (full-time equivalent) by manual excision to 117,600 per person by the methods described here. A summary of the benefits obtained in specific embodiments of the invention is shown in tables 1416. The mechanized processes described herein are also significantly more ergonomically less harmful in that they eliminate the repetitive motion typical of the manual excision process. In the case of cotton in which substantial numbers of explants may be required, the benefits are particularly significant.

[0026] To provide a clear and consistent understanding of the specification and claims, including the scope given to these terms, the following definitions are provided.

[0027] "Embryo" is part of a seed, consisting of precursor tissues (meristematic tissues) for the leaves, stalk and root, as well as one or more cotyledons. After the embryo starts to grow (germinate), it becomes a seed plant.

[0028] "Meristem" or "meristematic tissue" consists of undifferentiated cells, the meristematic cells, that differentiate to produce multiple plant structures, including stalk, roots, leaves, germ-line tissue and seeds. The meristematic cells are the targets for transformation in order to obtain transgenic plants.

[0029] "Explant" is a term used to refer to target material for transformation. Therefore, it is used interchangeably with "meristematic tissue" or "embryo" in the modalities in question.

[0030] "Chimeric plants" are plants that are composed of tissues that are not generically identical, that is, the plants will have only a part or fraction of their tissues transformed, while the remaining tissues are not genetically transformed.

[0031] "Germline transformation" occurs when the gene of interest is transformed into cells that generate pollen or ovum thereby producing seeds.

[0032] The seeds from which explants are to be prepared can be harvested from cotton cultivars of interest. Due to the fact that the methods described do not require embryogenesis or organogenesis, and the regeneration of cotton plants, the methods are suitable for use with many cultivars, even those known to have poor embryogenic potential. Therefore, in contrast to many previous methods, non-agronomically valued cultivars with good embryogenic potential, such as Coker 312, need not be used to obtain R0 starter transgenic plants. Adjustments can be made to parameters such as inhibition conditions (eg temperature and light intensity during seed soaking), roller spacings and sieve sizes to allow use with cotton seeds of various sizes. The seed for excision can be transgenic or non-transgenic. Therefore, the repeated transformation of an already transgenic cotton variety is contemplated.

[0033] Prior to imbibition, germination, and/or explant excision, seeds may be subjected to a sterilization step, as well as a separation step, to avoid microbial contamination, in order to remove seeds with a high degree of bacterial or fungal contamination, and also in order to remove seeds which may, for whatever reason, be unlikely to produce viable explant tissue for use with the present invention. Separation may be conducted, for example, based on parameters such as seed size, color, or density or other characteristics, including chemical composition characteristics. Floating a seed in an aqueous solution can be used to select seed for excision. Examples of separation methods may include the use of an automatic scale after grading by size or air grading of seed by using fans and/or vibrating gravity table to separate seeds by weight. Other separation techniques may also be employed, including separation by moisture content. A heat treatment can be carried out before soaking (eg 42-55°C). Osmotic seed conditioning can be used to control embryonic development before or after excision. Seeds may also be pretreated with certain chemical agents and/or environmental conditions before or during imbibition, germination, or excision to make the explants more transformable and regenerable after excision.

[0034] Soaking can be done in a clean, waterproof tank (sometimes called a soaking tank), such as a plastic or steel container that can hold the seeds. A typical capacity would be 1 to 12.5 kg of dry seeds, although other size containers can be used. The temperature and duration of seed soaking can range, for example, from about 15°C to about 40°C, such as about 20°C, 23°C, 24°C or 28°C. Temperature at the lower end of the range can be useful for longer duration inhibition. The duration can range from several hours to 14-48 hours, or several days. In certain embodiments, a soak period of about 18 hours may be used.

[0035] In specific modalities, excision is performed mechanically using rollers that crush the seeds applied to their faces, which can rotate contrary, in specific modalities. The distance between the rollers can be adjusted, based on the size of the seeds applied. In certain embodiments in which cottonseed is being crushed, the distances between rollers can be, for example, in the range of 2-4.5 mm (measured from the peaks between opposite rollers). The material of the rollers can be, for example, elastomeric or metallic. In certain embodiments, stainless steel rollers have been shown to retain beneficial running qualities even after repeated and prolonged use. For use with cotton seeds, secondary grooved rollers have been shown to efficiently grab and crush the seed with minimal damage to the seed fraction of meristematic explants. Excision can be performed on dehydrated cottonseed. The meristematic tissues obtained from them can then be rehydrated and transformed.

[0036] After excision, the invention also provides methods and apparatus for sorting to separate transformable meristematic explant material from explants, cotyledons, seed coats, and other damaged non-transformable fragments. The methods can be performed manually, or they can be partially or fully mechanized. In certain embodiments, the sorting process is substantially mechanized. For example, one or more sieving steps can be performed, using sieves of the appropriate size, based on the size of the seeds being crushed and the explants being isolated. The gross yield of the crushed seeds that have passed through the rollers can be fed through a series of separation screens such that unwanted large and small fragments are separated from the desired explant by size exclusion. The sieving sieves can be tilted to facilitate movement of plant material. The movement of material along or through the sieves (eg embryos and fragments) can be assisted, for example, by a flow of water, including water spray, or by a combination of sieves tilt and water spray. Vibration of the sieve surfaces can also aid in the movement of plant material. Continuous sieving can be performed using a rotary turner made from a sieve with an appropriately sized mesh. The sieving can be carried out effectively, for example, with a cottonseed material, using North American standard sieves such as: #8 (2.36 mm opening), #10 (2.0 mm opening), #1 16 (1.18 mm opening), and others as appropriate (e.g., sieves with elongated openings, such as 1/16" x 3/4", 1/18" x 3/4", 1/19" x 1/2", or 1/20" x 1/2"). Sieves with other opening sizes can be manufactured as needed, based on the size of the material being applied. The length of time for the sieving process and sieving vigor can also be adjusted to increase production volume and/or process throughput.

[0037] The apparatus may further include a water and seed containment system to help maintain sterility, as well as an ongoing clean system, allowing convenient maintenance and cleaning of the machine.

[0038] Other sieving methods may also be used, such as measuring the differential fluctuation in solutions of the explant material versus fragments. A fraction of material that floats in aqueous solution has been shown to be enriched for intact transformable explants. Combinations of these sieving methods can also be used. The fraction of material with transformable explants may comprise meristematic tissues and other tissues, such as parts of cotyledons. The explant may, however, contain at least some meristematic region such that typically the explant can produce a bud or bud within 12 weeks after the onset of appropriate growth conditions.

[0039] In some embodiments, excised meristematic material may be stored prior to commencing co-cultivation or other transformation procedure. Parameters that can be varied include temperature, duration, and storage medium, among others. For example, explants can be stored submerged in medium, for example, INO medium or degassed INO medium at 0°C or higher, for example, about 4-15°C. The explants can also be stored on filter paper moistened with INO, for example at 4°C. Medium components such as antibiotics, PE G 8000, and antioxidants can also be used. The storage duration can be in the range, for example, between one or more hours and overnight, or 1, 2, 3, 4 days or even 7 days. Storage can serve to adjust an embryo's developmental stage in relation to transformation time, as well as to aid in programming excision and transformation procedures, or to allow excised material to recover from the mechanical stress of excision. Wet excised meristematic explants can be dried for storage and then rehydrated for inoculation. Such drying can take place, for example, before or during sieving. Inoculation can be carried out, for example, for 10-120 minutes, during which time the explants are incubated in a vascolejador with an Agrobacterium suspension. The suspending medium may be INO, or other medium. After this inoculation step, the suspension is removed and the co-culture continues in INO, for example. Anti-apoptopic agents can be used during co-culture, including antipain (1-100 uM); 3-aminobenzamide ((5uM-4mM), Ac-DEVD-CHO (0.01-0.1uM) and Ac-YVAD-CMK (0.01-0.1uM), or other caspase inhibitor among others. Plant growth regulators and other agents that promote T-DNA transfer and plant regeneration can also be included to enhance plant transformation and regeneration. Sulphite can also be added to the co-culture medium to improve the plant tissue health. Antibiotics and antifungal agents, such as streptomycin, spectinomycin, nystatin, and thiabendazole, among others, can be used to supplement the coculture medium. Coculture can be conducted in the dark, or under appropriate lighting conditions to promote normal plastid development, such as in an illuminated Percival incubator with a photoperiod of 16 h light/8 h dark under a light intensity > 5 μ E, such as about 5 μ E to about 200 μ E. Such lighting conditions can promote the transfer evidence of Agrobacterium (Zambre et al., 2003).

[0040] In certain embodiments, excised and sorted tissues can be transformed with a heterologous gene of interest. Several methods have been developed to transfer genes into plant tissue, including high-velocity microinjection of microprojectiles, electroporation, direct DNA uptake, and bacterially mediated transformation. Bacteria known to mediate plant cell transformation include numerous species of rhizobiaceae, including, but not limited to, Agrobacterium spp., Sinorhizobium spp., Mesorhizobium spp., Rhizobium spp., and Bradyrhizobium spp. (e.g., Broothaerts et al., 2005; US Patent Application Publication No. 2007/0271627). The targets for such transformation were often undifferentiated callus tissue, although undifferentiated tissue has been used for transient and stable plant transformation, and may be in this case.

[0041] When drawing a vector for the transformation process, one or more genetic components are selected that are introduced into the cell or plant tissue. The genetic components may include any nucleic acid that is introduced into a plant cell or tissue using the method according to the invention. In one embodiment, the genetic components are incorporated into a DNA composition, such as a plasmid molecule or double-stranded recombinant vector that comprises at least one or more of the following types of genetic components: (a) a promoter that functions in cells plants to cause the production of an RNA sequence, (b) a structural DNA sequence that causes the production of an RNA sequence that encodes an agronomic utility product, and (c) an untranslated 3' DNA sequence that works in plant cells to cause the addition of polyadenylated nucleotides to the 3' end of the RNA sequence.

[0042] The vector may contain numerous genetic components to facilitate the transformation of the cell or plant tissue and regulate the expression of the sequence of structural nucleic acids. In the preferred embodiment, the genetic components are oriented to express an mRNA which is an optional embodiment and can be translated into a protein. Expression of a plant structural coding sequence (a gene, cDNA, synthetic DNA, or other DNA) that exists in a double-stranded form involves transcription of messenger RNA (mRNA) from one strand of DNA by the enzyme RNA polymerase and subsequent processing of the primary mRNA transcript within the nuclei. This splicing involves a 3' untranslated region that adds polyadenylated nucleotides to the 3' ends of the mRNA. Means for preparing plasmids or vectors that contain the desired genetic components are well known in the art.

[0043] Transcription of DNA into mRNA is regulated by a region of DNA commonly referred to as the "promoter". The promoter region contains a sequence of bases that signals RNA polymerase to associate with DNA and to initiate transcription into mRNA using one of the strands of DNA as a template to produce a corresponding complementary strand of RNA. Numerous promoters that are active in plant cells have been described in the literature. Such promoters would include, but are not limited to, the nopaline synthase (NOS) and octopine synthase (OCS) promoters that are carried on Ti plasmids of Agrobacterium tumefaciens, the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) promoters 19S and 35S and the scrofularia mosaic virus (FMV) 35S promoter, and the CaMV35S enhanced promoter (e35S). Several other plant gene promoters, which are regulated in response to environmental, hormonal, chemical and/or developmental signals, may also be used for heterologous gene expression in plant cells, including, for example, promoters regulated by (1) heat (Callis et al., 1988, (2) light (e.g., pea RbcS-3A promoter, Kuhlemeier et al., (1989); maize RbcS promoter, Schaffner et al., (1991); (3) hormones, such as abscisic acid (Marcotte et al., 1989, (4) wounding (eg, Wuni, Siebertz et al., 1989), or other signals or chemicals. Tissue-specific expression is also known.

[0044] As described below, it is preferred that the specific promoter selected is capable of causing sufficient expression to result in the production of an effective amount of the gene product of interest. Examples describing such promoters include, without limitation, US Patent No. 6,437,217 (RS81 promoter from corn), US Patent No. 5,641,876 (actin promoter from rice), US Patent No. 6,426,446 (RS324 promoter). of corn), US Patent No. 6,429,362 (PR-1 promoter of corn), US Patent No. 6,232,526 (A3 promoter of corn), US Patent No. 6,177,611 (constitutive promoters of corn), US Patents Nos. 5,322,938, 5,352,605, 5,359,142 and 5,530,196 (35S promoter), US Patent No. 6,433,252 (corn L3 oleosin promoter), US Patent No. 6,429,357 (actin promoter) 2 of rice as well as a rice actin 2 intron), US Patent No. 5,837,848 (root-specific promoter), US Patent No. 6,294,714 (light-inducible promoters), US Patent No. 6,140. 078 (salt-inducible promoters), US Patent No. 6,252,138 (pathogen-inducible promoters), US Patent No. 6,175,060 (phosphorus deficiency-inducible promoters), US Patent No. 6,635,806 (gamma- coixina), and patent application in serial US 09/757,089 (maize chloroplast aldolase promoter). Additional promoters that may find use are a nopaline synthase (NOS) promoter (Ebert et al., 1987), the octopine synthase (OCS) promoter (which is carried on Agrobacterium tumefaciens tumor-inducing plasmids), the Caulimoviruses such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., 1987), the CaMV 35S promoter (Odell et al., 1985), the scrofula mosaic virus 35S promoter (Walker et al., 1987; US Patent Nos. 6,051,753; 5,378,619), the sucrose synthase promoter (Yang et al., 1990), the R gene complex promoter (Chandler et al., 1989 ), and the chlorophyll a/b binding protein gene promoter, PCISV (US Patent No. 5,850,019), and AGRtu.nos promoters (GenBank Accession Number V00087; Depicker et al, 1982; Bevan et al., 1983).

[0045] Promoter hybrids can also be constructed to enhance transcriptional activity (US Patent No. 5,106,739), or to combine desired transcriptional activity, inducibility and tissue specificity or developmental specificity. Promoters that function in plants include, but are not limited to, promoters that are inducible, viral, synthetic, constitutive as described, and temporally regulated, spatially regulated, and spatially and also temporally regulated. Other promoters that are tissue-enhancing, tissue-specific, or developmentally regulated are also known in the art and anticipated to have utility in the practice of this invention.

[0046] The promoters used in the DNA constructs (ie, chimeric/recombinant plant genes) of the present invention can be modified, if desired, to affect their control traits. Promoters can be derived by means of linkage with operator regions, random or controlled mutagenesis, etc. In addition, promoters can be altered to contain multiple "enhancer sequences" to assist in elevating gene expression.

[0047] The mRNA produced by a DNA construct of the present invention may also contain an untranslated 5' leader sequence. This sequence can be derived from the promoter selected to express the gene and can be specifically modified so as to increase or decrease mRNA translation. The 5' untranslated regions can be obtained from viral RNA's, from appropriate eukaryotic genes, or from a synthetic gene sequence. Such "enhancer" sequences may be desirable to increase or alter the translation efficiency of the resulting mRNA. The present invention is not limited to constructs in which the untranslated region is derived from a 5' untranslated sequence accompanying the promoter sequence. Instead, the untranslated leader sequence can be derived from unrelated promoters or genes (see, for example, US Patent No. 5,362,865). Examples of untranslated leader sequences include corn and petunia heat shock protein leaders (US Patent No. 5,362,865), plant viral coat protein leaders, plant rubisco leaders, GmHsp (US Patent No. 5,659,122), PhDnaK (US Patent No. 5,362,865), AtAntl, TEV (Carrington and Freed, 1990), and AGRtu.nos (GenBank Accession No. V00087; Bevan et al., 1983). Other genetic components that serve to enhance expression or affect transcription or translation of a gene are also envisioned as genetic components.

[0048] The 3' untranslated region of chimeric constructs may contain a transcriptional terminator, or an element that has equivalent function, and a polyadenylation signal that functions in plants to cause the addition of polyadenylated nucleotides to the 3' end of the RNA. DNA sequences are referred to herein as transcription termination regions. The regions are necessary for efficient polyadenylation of transcribed messenger RNA (mRNA). RNA polymerase transcribes a coding DNA sequence through a site where polyadenylation occurs. Examples of suitable 3' regions are (1) the 3' transcribed, untranslated regions that contain the polyadenylation signal of Agrobacterium tumor-inducing plasmid (Ti) genes, such as the nopaline synthase gene ( NOS, Fraley et al., 1983), and (2) plant genes such as the soy storage protein genes and the small subunit of the ribulose-1, 5-bisphosphate carboxylase gene (ssRUBISCO). An example of a preferred 3' region is that of the pea ssRUBISCO E9 gene (European Patent Application No. 0385 962).

[0049] In one embodiment, the vector contains a gene selectable, or capable of being screened or marked. These genetic components are also referred to herein as functional genetic components, as they produce a product that serves a function in the identification of a transformed plant, or a product of agronomic utility. The DNA that serves as a selection or sorting device can function in a regenerable plant tissue to produce a compound that would later confer the plant tissue's resistance to an otherwise toxic compound. A number of marker genes capable of being screened or selected are known in these techniques and can be used in the present invention. Genes of interest for use as a gene capable of being selected, screened or marked would include, but are not limited to, gus, green fluorescent protein (gfp), luciferase (lux), genes that confer antibiotic tolerance such as kanamycin (Dekeyser). et al., 1989) or spectinomycin (e.g. aminoglycoside spectinomycin adenyltransferase (aadA); US Patent No. 5,217,902), genes encoding herbicide-tolerance enzymes such as glyphosate (e.g., 5-enoylpyruvylshikimate). -3-phosphate synthase (EPSPS): Della-Cioppa et al., 1987, US Patent No. 5,627,061, US Patent No. 5,633,435, US Patent No. 6,040,497, US Patent No. 5,094,945; WO Nos. 04074443, and WO 04009761; glyphosate oxidoreductase (GOX; US Patent No. 5,463,175); glyphosate decarboxylase (WO No. 05003362 and US Patent Application No. 2004/0177399; or glyphosate N-acetyltransferase ( GAT): Castle et al., US Patent Publication No. 2003/0083480), dalapon (e.g., dehI encoding acid 2,2-dichloropropionic dehalogenase that confers tolerance to 2,2-dichloropropionic acid (Dalapon; WO No. 9927116)), bromoxynil (haloarylnitrilase (Bxn) to confer bromoxynil tolerance (WO No. 8704181A1; US Patent No. 4,810,648; WO No. 8900193A)), sulfonyl herbicides (e.g. aceto -hydroxy acid synthase or acetolactate synthase that confers tolerance to acetolactate synthase inhibitors such as sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates and phthalide; (US Patent Nos. 6,225,105; 5,767,366; 4,761,373; 5,633 437; 6,613,963; 5,013,659; 5,141,870; 5,378,824; 5,605,011); encoding ALS, GST-II), bialaphos, or phosphinothricin or derivatives (e.g., phosphinothricin acetyltransferase (bar) which confers tolerance to phosphinothricin or glufosinate (US Patent Nos. 5,646,024, 5,561,236, 5,276,268; 5,637,489; 5,273,894; and EP Document No. 275,957), atrazine (encoding GST-III), dicamba ( dicamba monooxygenase; US Patent Application Publication Nos. 2003/0115626, 2003/0135879), or setoxodim (acetyl-coenzyme A carboxy modified lase to confer tolerance to cyclohexanedione (sethoxydim) and aryloxy-phenoxy-propionate (haloxyfop) (US Patent No. 6,414,222), among others. Other selection procedures can also be implemented, including positive selection mechanisms (eg, use of the E. coli manA gene, which allows growth in the presence of mannose), and dual selection (eg, using simultaneously 75-100 ppm of spectinomycin and 3-10 ppm glufosinate, or 75 ppm spectinomycin and 0.2-0.25 ppm dicamba) and would still fall within the scope of the present invention. The use of spectinomycin at a concentration of about 251.00 ppm, such as about 150 ppm, is also contemplated.

[0050] The present invention can be used with any plasmid or plant transformation vector, which contains a selectable or screenable marker and associated regulatory elements, as described, together with one or more nucleic acids expressed in a manner enough to confer a specific desirable trait. Examples of appropriate structural genes of agronomic interest envisaged by the present invention would include, but are not limited to, genes for disease, insect or pest tolerance, herbicide tolerance, genes for quality improvement such as yield, nutritional enhancement, environmental tolerance environment or stress, or any desirable changes in physiology, growth, development, morphology of plants, or plant product or products, including starch production (US Patent Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876 ; 6,476,295), production of modified oils (US Patent Nos. 6,444,876; 6,426,447; 6,380,462), high production of oils (US Patent Nos. 6,495,739; 5,608,149; 6,483,008 ; 6,476,295), modified fatty acid content (US Patent Nos. 6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538; 6,589,767; 6,537,750; 6,489 461; 6,459,018), high protein production (US Patent No. 6,380,466), fruit ripening (US Patent No. 5,512,4 66), increased animal and human nutrition (US Patent Nos. 6,723,837; 6,653,530; 6,541,259; 5,985,605; 6,171,640), biopolymers (US Patent Nos. RE37,543; 6,228,623; 5,958,745 and US Patent Publication No. 2003/0028917). In addition, resistance to environmental stress (US Patent No. 6,072,103), pharmaceutical peptides and secretable peptides (US Patent Nos. 6,812,379; 6,774,283; 6,140,075; 6,080,560), better processing traits ( US Patent No. 6,476,295), better digestibility (US Patent No. 6,531,648), low raffinose content (US Patent No. 6,166,292), industrial enzyme production (US Patent No. 5,543,576), better taste (Patent No. US No. 6,011,199), nitrogen fixation (US Patent No. 5,229,114), hybrid seed production (US Patent No. 5,689,041), fiber production (US Patent No. 6,576,818; 6,271. 443; 5,981,834; 5,869,720) and biofuel production (US Patent No. 5,998,700). Any of these or other genetic elements, methods, and transgenes can be used with the invention, as will be appreciated by those skilled in the art in light of the present invention.

[0051] Alternatively, DNA sequences of interest can affect these phenotypes by encoding an RNA molecule that causes targeted inhibition of the expression of an endogenous gene through gene silencing technologies, such as antisense-mediated mechanisms, co-suppression, RNAi including RNAmi (e.g., US Patent Application Publication No. 2006/0200878).

[0052] Exemplary nucleic acids that can be introduced by the methods encompassed by the present invention include, for example, DNA sequences or genes from other species, or even genes or sequences that originate or are present in the same species, but are incorporated into recipient cells by genetic engineering methods rather than classical breeding or breeding techniques. However, the term "exogenous" is intended to also include reference to genes that are not normally present in the cell being transformed or perhaps simply not present in form, structure, etc., as found in the transforming DNA segment or gene, or genes that are normally present even if desired, for example, have been overexpressed. Accordingly, the term "exogenous" gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in that cell. The type of DNA included in exogenous DNA may include DNA that is already present in the plant cell, DNA from another plant, DNA from a different organism, or externally generated DNA, such as a DNA sequence that contains an antisense message. of a gene, or a DNA sequence that encodes a synthetic or modified version of a gene.

[0053] In one embodiment, the transformation of plant tissue is performed by methods mediated by Agrobacterium or other Rhizobia, and the DNA sequences of interest are present in one or more T-DNA's (US Patent Nos. 6,265,638, 5,731). 179; US Patent Application Publication Nos. 2005/0183170; 2003/110532 ), or another sequence (eg, vector backbone) that is transformed into a plant cell. T-DNA's which may be ligated by RB and/or LB sequences, and may have one end sequence or two adjacent end sequences. Sequences that can be transferred into a plant cell may be present in a transformation vector in a bacterial strain that is being used for transformation. In another embodiment, the sequences may be present on separate transformation vectors in the bacterial strain. In yet another embodiment, the sequences can be found in separate bacterial cells or strains used together for transformation.

[0054] The DNA constructs used for transformation in the methods of the present invention also generically contain the plasmid backbone DNA segments, which provide the function of replication and selection with antibiotics in bacterial cells, for example an Escherichia origin of replication coli, such as ori322, an Agrobacterium origin of replication, such as oriV or oriRi, and a region coding for a selectable marker such as Spec/Strp encoding aminoglycoside adenyltransferase Tn7 (aadA) which confers resistance to spectinomycin or streptomycin, or a gentamicin selectable marker gene (Gm, Gent). For plant transformation, the host bacterial strain is often Agrobacterium tumefaciens ABI, C58, LBA4404, AGLO, AGL1, EHA101, and EHA105 carrying a plasmid that has a transfer function for the expression unit. Other strains known to those skilled in these plant transformation techniques may function in the present invention.

[0055] Bacterial mediated gene delivery (eg, Agrobacterium-mediated; US Patent Nos. 5,563,055; 5,591,616; 5,693,512; 5,824,877; 5,981,840) can be done in cells in the meristem from an embryo excised from a seed (eg, US Patent No. 6,384,301). During the co-culture step or after an antifungal and/or antibacterial compound can be used. Non-limiting examples of these compounds include Nystatin, PCNB (pentachloro-nitrobenzene), and thiabendazole, among others. Spectinomycin or streptomycin can also be used, for example, before, during co-culture, or after. In certain embodiments, the spectinomycin may be added during co-culture (e.g., 100 or 150 ppm, or up to 300-500 ppm, or up to 1000 ppm), and optionally is not present in the medium of later culture steps.

[0056] The meristematic region can be cultivated in the presence of a selection agent. The selective agent can be "pulsed"; that is, the concentration of the selective agent being used can be varied at different stages in the pre-culture, co-culture, or subsequent tissue culture process. In certain embodiments, the pulsed selective agent is an aminoglycoside such as spectinomycin. The result of this step is the termination or at least retardation of the growth of most cells within which the foreign genetic construct has not been distributed with the simultaneous formation of buds, which originate from a single cell or a small aggregate of cells, including a transformed meristematic cell. The resulting transformed shoots and plants may be chimeric (e.g., periclinal chimeras) or they may be clonally derived. However, after a phenotype-positive seedling is identified, in certain modalities the continued use of selective agent(s), also called "secondary selection", can be avoided. Therefore, the resulting plants may, in this case, be chimeric with untransformed root tissue that grew in the absence of selection, even though the elongated shoot tissue, coming from a meristem that came into contact with a heterologous nucleic acid, in whether or not it may be chimerical. This avoidance of secondary selection can save time, labor, and can also result in significant savings in US No. of culture medium and vessels. The meristem can be cultivated in the presence of a selection agent, including, but not limited to, auxin-like herbicides such as dicamba, 2,4-D, or MCPA, glufosinate, glyphosate, imidazolinone class herbicides, inhibitors acetolactate synthase, protoporphyrinogen oxidase inhibitors, and hydroxy-phenyl-pyruvate-dioxygenase inhibitors, neomycin, kanamycin, paramomycin, G418, aminoglycosides, spectinomycin, streptomycin, hygromycin B, bleomycin, phleomycin, sulfonamides, streptothricin, chloramphenicol, methotrexate , 2-deoxyglucose, betaine-aldehyde, S-aminoethyl-L-cysteine, 4-methyl-tryptophan, D-xylose, D-mannose, benzyladenine-N-3-glucuronidase. Examples of various selectable markers and genes providing resistance against them are described in Miki and McHugh, 2004.

[0057] In light of this descriptive report, numerous other selectable or screenable marker genes, regulatory elements, and other sequences of interest will become apparent to those skilled in these techniques. Therefore, the foregoing discussion is intended to be exemplary and not exhaustive.

[0058] The use of such selective agents can facilitate the recovery of transformed germline cells in the case of chimeric plants of the R0 generation, such that a fully transformed R1 seed is produced; i.e. "chemical pruning", or at least growth inhibition of untransformed tissues of chimeric plant selections of the R0 generation for growth and development of transformed tissues that include transformed germ-line tissues capable of producing fully transformed plants in one generation subsequent. This can allow for simplified tissue culture and plant regeneration, for example, eliminating embryogenesis, making the process faster, less costly, and applicable to a wider range of cotton genotypes, including better cultivars that are generally difficult to transform. , as well as to other plants for which embryogenesis and regeneration procedures are not well developed, if at all. In certain embodiments, germline tissue may be selected within a chimeric plant or plant part to produce transformed germline tissue, as noted below.

[0059] Alternatively, markers capable of being screened or judged can be used to identify transgenic sectors and/or plants. Exemplary markers are known and include β-glucuronidase (GUS) which encodes an enzyme for various chromogenic substrates (Jefferson et al., 1987a; Jefferson et al., 1987b); an R locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al., 1988); a β-lactamase gene (Sutcliffe et al., 1978); a gene encoding an enzyme for the various chromogenic substrates are known (eg PADAC, a chromogenic cephalosporin); a luciferase gene (Ow et al., 1986); an xy1E gene (Zukowsky et al., 1983) that encodes a catechol dioxygenase that can convert chromogenic catechols; an β-amylase gene (Ikatu et al., 1990); a tyrosinase gene (Katz et al., 1983) that encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which, in turn, condenses to melanin; green fluorescent protein (Elliot et al., 1999) and a β-galactosidase.

[0060] As is known in these techniques, other methods for plant transformation can be used, for example as described by Miki et al., (1993), including the use of microprojectile bombardment (for example, US Patent No. 5,914 451; McCabe et al., 1991; US Patent Nos. 5,015,580; 5,550,318; 5,538,880).

[0061] Various tissue culture media are known which, when properly supplemented, support tissue growth and development, including the formation of mature plants from excised meristems. These tissue culture media can be purchased as a commercial preparation or specially prepared, and modified by those skilled in the art. Examples of such means include, but are not limited to, those described by Murashige and Skoog, (1962); Chu et al., (1975); Linsmaier and Skoog, (1965); Uchimiya and Murashige, (1962); Gamborg et al., (1968); Duncan et al., (1985); McCown and Lloyd, (1981); Nitsch and Nitsch (1969); and Schenk and Hildebrandt, (1972), or derivations from these appropriately supplemented means. Those skilled in these techniques are aware that media and media supplements such as nutrients and growth regulators for use in transformation and regeneration are usually optimized for the target crop or target variety of interest. Tissue culture media may be supplemented with carbohydrates such as, but not limited to, glucose, sucrose, maltose, mannose, fructose, lactose, galactose, dextrose, or carbohydrate ratios. Reagents are commercially available and can be purchased from numerous suppliers (see, for example, Sigma Chemical Co., St. Louis, MO; and PhytoTechnology Laboratories, Shawnee Mission, KS). An elevated temperature step (eg 17 or 3-5 days of culture at 35°C) can be performed at the beginning of tissue culture, after co-culture. Explants can also be grown, for example, during co-culture and selection, under lighting conditions that allow normal plastid development. Therefore, explants can be grown under a light intensity of 5-200 μ Einsteins, such as 5-130 μ Einsteins, with a photoperiod of 16 light/8 dark hours.

[0062] Transgenic plants can be regenerated from a transformed plant cell by methods and compositions described herein. When putative transgenic seedlings are identified in the culture, they can be transplanted. Post-transplant growth media may include soil, or a soil-free medium in a pot or plug, such as an OASIS plug, Fertiss™ plug, or Elle pot. A plant growth regulator such as Mepiquat pentaborate can be used to reduce plant size and facilitate handling of large numbers of plants. A transgenic plant formed using Agrobacterium transformation methods typically (but not always) contains a single recombinant DNA sequence within a chromosome and is referred to as a transgenic episode. Such transgenic plants may be referred to as being heterozygous for the inserted exogenous sequence. A transgenic plant homozygote with respect to a transgene can be obtained by sexual reproduction (self-crossing) of an independently segregating transgenic plant that contains a single exogenous gene sequence for itself, for example an R0 plant, to produce an R1 seed. A quarter of the R1 seed produced will be homozygous for the transgene. The germinating R1 seed results in plants that can be tested for zygosity, typically using an SNP assay or a thermal amplification assay that allows the distinction between heterozygotes and homozygotes (ie, a zygosity assay).

[0063] To confirm the presence of exogenous DNA or "transgene(s)" in transgenic plants, a series of assays can be performed. Such assays include, for example, "molecular biology" assays, such as Southern and Northern blotting and PCR™; "biochemical" assays, such as detection of the presence of a protein product, for example, by immunological means (ELISAs and Western blots) or by enzymatic function (for example, GUS assay); pollen histochemistry; plant part assays, such as leaf or root assays; and also analyzing the phenotype of the total regenerated plant.

[0064] Once a transgene has been introduced into a plant, this gene can be introduced into any plant sexually compatible with the first plant by crossing, without the need to always directly transform the second plant. Therefore, as used herein, the term "progeny" denotes the offspring of any generation of a parental plant prepared in accordance with the present invention, where the progeny comprise a selected DNA construct. A "transgenic plant" can thus be of any generation. The "crossing" of a plant to produce a plant lineage that has one or more transgenes or alleles added relative to an originating plant lineage is defined as the techniques that result in a specific sequence being introduced into a plant lineage. by crossing a starter line with a donor plant lineage that comprises a transgene or allele. To achieve this one could, for example, carry out the following steps: (a) planting seeds from the first (starter line) and second (donor plant line that comprises a desired transgene or allele) parent plants; (b) growing the seeds of the first and second parent plants to give flower-bearing plants; (c) pollinating a flower from the first parent plant with pollen from the second parent plant; and (d) harvesting seeds produced on the parent plant bearing the fertilized flower.

[0065] The present invention also provides parts or the plant produced by the methods of the present invention. The plant parts, without limitation, include fruit, seed, endosperm, ovule, pollen, leaf, stalk, and roots. In a preferred embodiment of the present invention, the plant part is a seed.

Examples

[0066] Those skilled in these techniques should appreciate the many advantages of the methods and compositions provided by the present invention. The following examples are included to demonstrate preferred embodiments of the invention. Those skilled in the art should appreciate that the techniques described in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and as such, can be considered to constitute preferred modes for its practice. However, those skilled in the art should, in light of the present specification, appreciate that many changes can be made to the specific embodiments described and still obtain a similar or similar result without departing from the spirit and scope of the invention. All references cited herein are incorporated herein by reference to the extent that they supplement, explain, provide background on, or set forth the methodology, techniques, or compositions employed herein.

Example 1 Sterilization, Hydration, and Excision of Cottonseeds, and Recovery of Meristem Explants. A. Cottonseed Hydration Sterilization

[0067] Cottonseeds were mechanically processed to excise and isolate their meristematic tissues. To obtain transformable meristematic explant material, cottonseeds (e.g., genotypes STN474 (Stoneville Pedigreed Seed Co., Stoneville, MS), Delta Pearl (Delta and Pine Land Co., Scott, MS), DP5415, DP393, 00S04 (Delta and Pine Land Co.), SureGrow501 or SureGrow747 (Sure Grow Cotton Seed Company, Maricopa, AZ) were processed as follows to separate the embryo comprising meristematic tissues from the seed coat and cotyledon(s). Cotton seeds were removed from storage at 4°C or -20°C and brought to room temperature. The seeds were weighed, placed in a sterile imbibition unit, and surface sterilized in 50% Clorox (sodium hypochlorite) for 5 min. The seeds are then rinsed 3 times with sterilized distilled water and hydrated in a liquid hydration medium (CSM) at 28°C in the dark for about 18 h (range 14 to 42 h). Alternatively, the soaking temperature may be lower, for example about 23°C. CSM medium contained 200 mg/L carbenicillin (PhytoTechnology Laboratories, Shawnee Mission, KS), 125 mg/L cefotaxime (Midwest Scientific, St. Louis, MO), 30 mg/L BRAVO 75 (Carlin, Milwaukee, WI) and 30 mg/L of Captan 50 (Carlin). Other solutions have also been used successfully to hydrate cottonseeds, including sterile demineralized water or water containing a weak concentration of bleach (typically 50 to 1000 ppm sodium hypochlorite). After hydration, the seeds can be used immediately, or stored at refrigerated temperatures for up to a week before further processing.

B. Crushing of Cotton Seeds

[0068] A current model of excision machine is illustrated in Figure 1. The surface-sterilized hydrated seeds were loaded without guidance into the excision machine in batches or continuously through a feeder located on top of the machine (Figure 1B, 1C). Mechanical excision of embryos was performed using the excision machine in a dedicated clean room. The excision machine contains 1 pair of steel rollers to crush the seeds. The steel rollers (figure 1D, 1E) were modified from those used for excision of soybean meristems (e.g. US Patent Application No. 2005/0005321) changing the material from elastomer to stainless steel, reducing the number of rollers in pairs of 3 to 1, and adding grooves along the geometric axis of the rollers to better grip the cotton seeds and more efficiently crush the seeds. A comparison of the modified steel rollers with the previously developed elastomer rollers used in soybean embryonic shaft excision is presented in Table 1. "Quality explants" means explants with viable meristematic tissue (ie, useful as transformation targets). Table 1. Comparison between modified steel rollers and elastomer rollers.

Tabela 3. Efeito da velocidade dos roletes sobre o rendimento de explantes.

Tabela 4. Efeito da vazão de água sobre a recuperação de explantes.

[0069] Test results on machine parameters such as roller clearance distance and roller speed are summarized in Tables 2-4. The following example adjustments have shown to work well, among others: 2.5 mm roller spacing distance; right roller turning clockwise at setting 40 (arbitrary speed adjustment number on dial); left roller turning counterclockwise at setting 80; water flow rate of 10 L/min.Table 2. Effect of distance between rollers on explant yield.

Table 3. Effect of roller speed on explant yield.

Table 4. Effect of water flow on explant recovery.

Example 2 Meristematic Explant Separation by Sieving.

[0070] After the seed material passed through the roller mechanism), the crushed seed mixture (figure 2) was collected by a sieve which allows water to escape but retains crushed seed. The effect of roller clearance on the condition of meristematic tissues from crushed seeds is also illustrated in Figure 2. The retained material was then sieved to remove debris and enrich for potentially regenerable and transformable explants comprising meristematic tissues. One method of separating meristematic seed coat explants and cotyledon material from the coarsely crushed seed was by mechanized or manual sieving, illustrated in Figure 2. Various materials were tested in the construction of sieves, including carbon steel sieves and steel sieves. stainless steel with aluminum, wood or steel sides. A "V" shaped cross section of the sieve proved to be easy to fabricate and effective in retaining explants while allowing smaller debris to pass through (figure 3). The meristematic explants of the crushed seeds could also be efficiently separated by a two-step sieving method, which can be performed mechanically or manually. First, the crushed seeds (a mixture of seed coats, cotyledons and embryos) were passed through a size 8 sieve, which retained the large debris (seed coat and cotyledon), while allowing small debris and explants pass through.

[0071] The resulting mixture was then sieved again using a second stainless steel sieve. Various aperture sizes in the range between, for example, 1/16" x 3/8" and 1/24" x 1/2" were tested. An opening size of 1/18" x 3/4" or 1/19" x 3/4" gave the best explant purity with minimal additional loss in explant recovery. Therefore, sieves with an opening size of 1/18" x 3/4" or 1/19" x 3/4" were chosen for further use in the second sieving step. This parameter can be adjusted based on seed size and expected explant size. The second sieve retained explants and some larger debris, but allowed the small debris to pass through. Table 5 indicates a comparison of the resulting material after the two sieving steps, involving a mechanized or manual procedure. In the first case, an initial manual sieving step was followed by a machine sieving step. In the second case, both sieving steps were performed by machine. Both sieving and manual sieving can separate meristematic explants from other debris. Machine sieving appears to be more efficient than manual sieving in producing explants useful for transformation.Table 5. Recovery of explants using manual and/or mechanized sieving.

[0072] Other parameters that can be varied during the sieving process include the amount of seed (e.g. grams of seed per batch), vigor and length of the sieving process (e.g. in minutes), leading to better purity and yield of transformable material, measured by the number of "quality explants" per gram of material sieved, or by the number of "quality explants" recovered per minute of sieving. The term "quality explants" means explants with meristems useful as transformation targets. It has been observed that the highest process productivity (but not necessarily the number of explants) is achieved when a large batch of crushed seeds is sieved for a short time. Although the absolute number of "quality explants" can be reduced somewhat in this case, the time savings more than make up for this. Greater purity of explants can also be achieved. This assumes, however, that the amount of crushed seed available is not limiting, due to the relatively small contribution of excision time to the overall extent of the process to obtain transformable cotton explant tissue. Therefore, if the amount of seed is limiting, the sieving parameters can be adjusted accordingly. Quality explants prepared using the automated process described are ready to be transformed, or can be stored prior to use at refrigerated temperatures, or up to 28°C before use. Storage at these temperatures can maintain quality and processing capacity for at least 1 week. Refrigeration is preferred for longer storage.

Example 3 Additional Enrichment of Recovered Embryonic Explants

Tabela 7. Enriquecimento da Pureza de Explantes por Intermédio do Uso de Etapa de Peneiração com Flutuação

[0073] The explants recovered from the above example still contained some debris. The explants were further purified by a flotation method. The method was based on the observation that most recently sieved explants, but not most debris, are able to float to the surface of fresh sterile demineralized water. This step removed additional debris, improving the purity of the explants (eg, Figure 2) and as indicated in Tables 6-7. Flotation separation (i.e., discarding the settling fraction) may result in some loss of transformable explants, as both floating and settling fractions contained transformable explants, as concluded by subsequent transient transformation with Agrobacterium and GUS staining of explant material from each explant fraction. However, the purity of the explants of the floating fraction is considerably improved compared to the material without the flotation step.Table 6. Enrichment of Explants by Flotation.

Table 7. Enrichment of Explant Purity through the Use of Floating Sieving Stage

Example 4 Additional Process Improvements.

[0074] To further improve the efficiency of the process, further modifications of the excision unit ("excisioner") are carried out. For example, a water and seed containment tray is added to ensure seed containment (figure 4). The tray can be, for example, a rectangular stainless steel tray located under the machine outlet, with side walls on three of the four sides of the tray. The fourth side contains means for affixing a disposable mesh bag that retains seeds and debris while allowing water to escape.

[0075] A "clean in place" system can also be added to the explant production system, to simplify equipment cleaning, and reduce the possibility of workplace accidents and sample contamination. Cleaning procedures involve removing debris, using a stream of sterile water and forceps if necessary, and then pumping out a sanitizing solution (e.g., peracetic acid, such as MINNCARE (Minntech, Minneapolis, MN)) or gas. through the machine for 10 minutes at about 10 L/min. The sanitizing solution is extruded from the machine by applying sterile pressurized air. The clean machine is kept, between uses, under positive pressure of sterile air. An automated cleaning-in-place system was designed. The mechanism controls the cleaning process as follows: first, a sanitizing agent (e.g. 1% MINNCARE or similar) is pumped through the equipment for a set period of time (e.g. 10 min or as recommended by the appliance manufacturer). sanitizing agent). The agent flow is then discontinued, and sterile pressurized air is purged through the equipment to remove remaining liquid. Finally, sterile positive pressure air is applied as a sterile storage medium for the equipment between uses. In the case of the sieve, the removable parts are sent to an autoclave, while the stationary parts can be sprayed with 1% MINNCARE solution or another sterilizing solution.

Example 5 Development of a Transformation Method for Cotton Using Excised Embryos.

[0076] This example describes Agrobacterium-mediated transformation of cotton using mechanically excised embryos.

A. Plant Expression Constructs for Transformation.

[0077] Agrobacterium tumefaciens transformation vectors were constructed using standard molecular biology techniques known to those skilled in these techniques. The following transform constructs were used:

[0078] (1) pMON96959: which contains the uidA gene under the control of an enhanced CaMV.35S promoter (US Patent Nos. 5,322,938; 5,352,605; 5,359,142; and 5,530,1960, a sequence- 35S leader, and a 3' untranslated region of the nopaline synthase gene from Agrobacterium tumefaciens (Genbank Accession No. E01312); and the selectable marker Dicamba Monooxygenase (DMO) from Pseudomonas maltophilia (US Patent No. 7,022). .896) attached to the chloroplast by a chloroplast transit peptide and the first 24 amino acids of the mature pea ribulose 1,5-bis-phosphate carboxylase (rbcS) small subunit (rbcS) protein. The BMD gene cassette contains a enhanced promoter for the full-length peanut chlorotic streak virus (PCISV.Flt) transcript, a leader sequence from the 5' untranslated region of the tobacco Etch virus RNA genome (TEV Carrington and Freed, 1990), and a 3' untranslated region of pea rbcS2 (Coruzzi et al., 1984).

[0079] (2) pMON96999: which contains the same uidA cassette described for pMON96959, but a different selectable marker, an aadA gene (e.g., US Patent No. 5,217,902) to confer spectinomycin resistance. The aadA adenylyltransferase gene product was targeted to the chloroplast by a chloroplast transit peptide from Arabidopsis EPSPS (ShkG-CTP2 Klee et al., 1987.), and came under the control of the promoter for the Ara elongation factor. - bidopsis EF-1alpha (Tsf1; US Patent Application No. 2005/0022261) with an FMV-35S enhancer, a Tsf1 leader (exon 1), a Tsf1 intron, and a 3' untranslated region of pea rbcS2 .

[0080] (3) pMON102514: which includes the DMO and bar genes for use in single or double selection schemes with dicamba and/or glufosinate. The DMO gene cassette comprises an enhanced promoter of the full-length peanut stria chlorotic virus (PCLSV.Flt; US Patent No. 5,850,019) transcript, the TEV leader sequence, the 3' untranslated region of Sea-island cotton E6 fiber protein gene, and Arabidopsis ShkG-CTP2 chloroplast transit peptide to assign BMD to chloroplast. The bar gene cassette contains an enhanced CaMV.35S promoter, an Hsp70 petunia leader (US Patent No. 5,362,865), a bar gene, and an Agrobacteriumr nos terminator.

[0081] (4) pMON107303: which contains the uidA gene under the control of an enhanced CaMV.35S promoter, a 35S leader sequence, and a 3' untranslated region of the Agrobacterium tumefaciens nopaline synthase gene; and the bar gene cassette contains the same regulatory elements, but without the chloroplast transit peptide sequence as the aadA cassette in pMON96999.

[0082] The examples of transformation constructs described above all contained a single T-DNA for transgene integration into the plant genome. Transformation vectors with 2 T-DNAs were also used with this invention. An example of such vectors is described below:

[0083] (5) pMON107375: carrying 2 T-DNAs, both containing 2 marker genes, in a head-to-tail orientation. The first T-DNA includes an aadA and a uidA cassette. The aadA adenylyltransferase gene product is targeted to the chloroplast by a chloroplast transit peptide from Arabidopsis EPSPS (ShkG-CTP2, Klee et al., 1987), and the gene is under the control of the promoter for the elongation factor. from Arabidopsis EF-1alpha (Tsf1; US Patent Application No. 2005/0022261) with an FMV-35S enhancer, a Tsf1 leader (exon 1), a Tsf1 intron, and a 3' untranslated region of pea rbcS2 . The uidA gene is under the control of an enhanced CaMV.35S promoter, and a 3' untranslated region of the Agrobacterium tumefaciens nopaline synthase gene. The second T-DNA consists of a DMO and bar expression cassette. The DMO gene cassette comprises an enhanced promoter for the full-length peanut stria chlorotic virus transcript (PCLSV.Flt, US Patent No. 5,850,019), the TEV leader sequence, the 3' non- translated from the Sea-island cotton E6 fiber protein gene, and an Arabidopsis ShkG-CTP2 chloroplast transit peptide to assign BMD to the chloroplast. The BMD gene was codon-optimized for enhanced dicotyledon expression. The bar gene cassette contains an enhanced CaMV.35S promoter, an Hsp70 petunia leader (US Patent No. 5,362,865), a bar gene, and an Agrobacterium nos terminator. pMON107353 (oriV), which is similar to pMON107375 (oriRi), except for the origin of replication, was also used extensively in the studies described here.

[0084] The use of this construct and derived episodes of interest demonstrate the utility of chemical pruning using glufosinate as a means to identify transgenic plants and/or positively transformed sectors in chimeric plants, and chemically eliminating negative sectors.

B. Preparation of Agrobacterium Cells.

[0085] The Agrobacterium C58 strain that contains a binary vector with one or two plant expression cassettes, as described above, was inoculated, from a glycerin input, into a liquid LB medium (10 g/L of sodium chloride, 5 g/L yeast extract, 10 g/L bacto-tryptone), containing 75 mg/mL spectinomycin and 50 mg/mL kanamycin. The liquid culture was allowed to grow at 28°C at 200 rpm in a rotary vase overnight. After the optical density (OD660) of the overnight culture reached the target range of 0.4-1.2, the bacterial culture was centrifuged at 3500 rpm for approximately 20-25 min until the cells were pelleted.

[0086] After removing the supernatant, the pellet was resuspended in 10 ml of an inoculation medium (INO, table 8), and diluted further and adjusted to about 0.28-0.32 in OD660. A series of transient expression studies of GUS indicated that an OD660 of 0.6-0.8 of the inoculum produced a comparatively higher proportion of meristematic transformation and transgene expression.Table 8. Inoculation Medium Composition (INO).

[0087] The pH of the Agrobacterium preparation medium (growth medium, resuspension and co-culture) has been shown to affect the transformation efficiency of excised cotton meristems. A pH of about 5.3 gave the highest subsequent proportion of "high quality" stable transformation episodes, although media with another pH between 5.0 and 10.0 can be used. "High quality" episodes refer to transgenic episodes that contain a large area of stable expression of GUS, for example in one leaf, including the central vein, or stable expression of GUS in all or multiple leaves, as demonstrated by assay histochemistry of GUS. Such expression patterns have been shown to be most likely correlated with the L1, L2, or L3 cell layer of meristematic tissue (eg, periclinal transformation), which may result in germline transformation, or suggest single cell origin of the buds.

C. Wounding, Inoculation and Explant Coculture.

[0088] The explants of examples 1 or 2 were rinsed in sterile water. About 1-60 g, e.g. 30 g, of explants were placed inside the top (upside-down) part of a Plantcon® container (MP Biomedicals, Solon, OH), and then approximately 50 mL was added. of the prepared Agrobacterium suspension, enough to cover the explants. After the Plantcon® was closed, it was inserted into an appropriately sized holder, which was placed inside an ultrasound device (e.g., L&R Ultrasonics QS140; L&R Manufacturing Co., Kearny, NJ; or an ultrasound device). ultrasound Honda W113, Honda, Denshi Japan). The ultrasound apparatus was filled with about 2 L of 0.1% Triton® (e.g. Sigma 526-36-23; Sigma Chemical Co, St. Louis, MO). After up to 5 min of ultrasound application, the Plantcon® was firmly affixed in a squeegee at 65-70 rpm or higher for 10 min for incubation. After inoculation, the Agrobacterium inoculum was removed from the Plantcon®. About 2 g of the inoculated explant tissue was transferred to a new Plantcon® containing sterile filter paper and 5 mL of INO, and the explants were spread over the surface of the medium to prevent clumping. INO medium was also supplemented with plant growth regulators such as gibberellins (GA3), auxins (e.g. NAA, IBA, IAA, 2,4-D, dicamba, etc.), cytokinins (e.g. BAP, thidiazuron, dikegulac, kinetin, etc.), and/or antifungal or antibacterial agents such as nystatin, or thiabendazole (TBZ). The Plantcon® containing inoculated explants was placed inside a Percival incubator for co-cultivation at approximately 22-28°C and a 16-hour light photoperiod (light intensity >5 μE, e.g. between about 5 μE and 200 μE) for 2-5 days. In certain studies, spectinomycin (50-100 ppm) was included in the co-culture medium. As illustrated in Figure 8, this led to enhanced TF.

D. Selection and Identification of Transgenic Episodes by the Use of Spectinomycin or Another Selective Agent in the Tissue Culture Medium.

[0089] This example describes selection with the antibiotic spectinomycin. However, analogous methods can be performed using other selection agents such as kanamycin, streptomycin, G418, paromomycin, glufosinate, glyphosate, hygromycin B, imidazolinone, and dicamba, or a combination of any of these or similar selection agents. A marker capable of being screened, such as GUS, CrtB, or a yeast ATP-dependent phosphofructokinase (ATP PFK), may also be employed.

[0090] After co-culture, the explants were removed and implanted or layered on the surface of a semi-solid WPM medium (Table 9) in Plantcon® containers supplemented with 200 mg/L cefotaxime, 200 mg/L carbenicillin and 25 -200 mg/L spectinomycin with or without plant growth regulators (such as BAP, tidiazuron, GA3 or dikegulac) as needed to stimulate multiple shoot formation. Typically, ~25 explants were placed within each container. The results indicate that plating on the surface of explants was at least as effective as implantation (Table 10). Plating the inoculated explants on the surface of a WPM liquid medium (Table 9, but without the AGARGEL gelling agent) was also tested to improve efficiency and to reduce the ergonomic burden of implanting explants into the medium. Plating on the surface of explants on liquid culture substrates (filter paper, polyester felt and other cloths, and other permeable and semi-permeable membranes were also successfully tested. The explants were allowed to develop on the selection medium at 28°C). °C (daily and seasonal variations from 22-33 °C) with a photoperiod of typically 16 h of light, although 24 h of light is also effective to produce transgenic cotton plants, and a light intensity of 5-200 μ Einsteins , typically 70-130°C, for about 4-8 weeks. An initial temperature of 35°C for the first 3-5 days during selection was also tested, before the explants were moved to 28°C. culture at 35°C has been shown to be beneficial (see, for example, example 9).

[0091] Regenerating explants with healthy green shoot growth (eg, figure 5) were subsequently transferred into fresh selection medium for continued growth (second selection step). In contrast, explants lacking a gene that confers resistance to spectinomycin produced bleached shoots or primordia when developed with spectinomycin, which could be easily identified. After 1-4 weeks, seedlings with healthy looking green shoots were transferred and implanted in Cotton Rooting Medium (CRM) (Table 11) for rooting. The rooting medium may comprise spectinomycin or streptomycin, or selection may be discontinued during the rooting step. The rooted plants were then taken to a greenhouse for continued growth and further analysis.

Tabela 10. Comparação de implantação de explante e plaqueamento superficial.

[0092] From the infection with Agrobacterium after excision and enrichment of the explants, the explants went through a selection process. The selected transgenic episodes then grew to give a mature plant. The transgenic R1 seed can be harvested for about 24 weeks after the start of a transformation experiment (e.g. co-culture with Agrobacterium), representing significant time and labor savings compared to a typical transformation and regeneration strategy. of cotton plants employing embryogenesis. Table 12 is illustrative of selection agents and effective rates tested. Table 9. Composition of Modified WPM Supplemented with Antibiotics.

Table 10. Comparison of explant implantation and surface plating.

[0093] Phenotype-positives: demonstrating visible phenotype indicative of resistance to the selective agent used (eg, greens appearing healthy growth properly formed on selection with spectinomycin as distinct from malformed bleached and necrotic non-transgenic phenotype-negative tissue);

Tabela 12. Agentes de seleção e suas taxas eficazes.

[0094] Quasi-periclinal transformation: a large area of 50% or more cut edge) of GUS expression in a leaf including the central vein, or stable GUS expression in multiple leaves, as tested by histochemical staining for GUS. Table 11. CRM Composition.

Table 12. Selection agents and their effective rates.

[0095] Spectinomycin served as a useful visual marker for early identification of transformation. Non-transformed tissues usually appeared bleached and often malformed upon selection with spectinomycin, while transformed tissues were green and properly developing (figure 5). The transformed nature of the green tissue was confirmed by GUS expression after about 4-8 weeks on the selection medium. Therefore, using spectinomycin as a selection agent predates the labor-intensive and time-consuming GUS trials often used in meristem transformation systems and provides the advantage of significantly reducing the labor involved to produce transgenic plants.

E. Selection and Identification of Transgenic Episodes by the Use of Chemical Pruning or Herbicide Spraying.

[0096] Chemical pruning by herbicide spray, ie, killing untransformed tissue, was also tested on plants that were transformed with transgenes for dicamba and glufosinate tolerance. The plants were placed in a special spray chamber, and hand sprayed with commercial herbicide formulations such as LIBERTY at 100-3000 ppm, and CLARITY at 1003,000 ppm. The plants were sprayed until runny, allowed to dry for about 2 hours and then taken back to the greenhouse. Non-transgenic tissues were usually killed or their growth was suppressed by the spray, while tissues expressing transgenes continued to grow, allowing easy identification of transgenic tissues. CLARITY and LIBERTY sprays often resulted in the development of side branches, which became the main growth, leading to an R1 seed comprising a transgene.

[0097] Similarly, herbicide sprays have been used to select transgenic plants in the absence of a selection agent, or after inefficient tissue culture selection. Regenerated episodes of unknown transgenic status after inoculation with a plasmid containing a herbicide tolerance gene were sprayed with an optimized dose of selective agent (eg, 1000 ppm glufosinate, to obtain the resistance phenotype.

F. Selection and Identification of Transgenic Episodes by the Use of an Analytical Assay.

[0098] Plants transformed with a plasmid that contained a visual marker gene such as uidA can be identified by histochemical assays of leaves, pollen, or other tissues, as described in WO No. 9215675; US Patent No. 5,164,310; McCabe and Martinell, 1993. Real-time PCR can also be used.

Example 6 Characterization of Transgenic Episodes Obtained from Mechanically Excised Cotton Explants.

[0099] This section describes the characterization of transgenic episodes. Episode No GH_A24519 is used as an example. Seeds (cotton cv. STN474) were superficially sterilized, rinsed, and soaked in CSM medium for about 16 hours at 28°C. The soaking seeds were then machine excised using steel rollers, set at a spacing of 2 to 3 mm (measured by the peaks of the opposing rollers, determined to be appropriate for this batch of STN474 seed), and sieved through a two-step sieving process (manual sieving with a #10 sieve, then automated sieving), and prepared and inoculated with Agrobacterium containing pMON96959, as described, for example, in examples 1-5. After co-culture, explants were implanted into solid WPM medium supplemented with 100 mg/L cefotaxime and 0.3 mg/L dicamba in 25 embryos by Plantcon®. The explants were allowed to grow on this selective medium for about one month at 28°C with a 16-hour light photoperiod. The GUS trials were performed one month later and several promising plants including GH_A24159 were transferred to CRM for root development. GUS assays to test for transformation were performed again 3 weeks later, and all sampled leaves showed a high level of GUS expression. A cross-sectional GUS histochemical assay showed GUS expression in petiole vascular tissues. The first flower of plant R0 also showed GUS expression in pollen, petal, sepal, and anthers.

[00100] Molecular analyzes were also performed to confirm the transformation. PCR results of leaf samples indicated the presence of BMD sequence. Southern blot hybridization analysis of the R0 plant confirmed the presence of at least 2 copies of the BMD transgene in sampled leaves (eg, lanes 2-4 of figure 6). Western blot analysis further demonstrated the expression of the DMO protein (Figure 7). The seedlings of the R0 plant were shown to be resistant to the spray of CLARITY at 200 ppm. To speed up the analysis of R1, 16-day-old immature embryos from the first flower were excised and studied. Eight stained for GUS activity, with six demonstrating GUS expression in all tissues, suggesting a 3:1 Mendelian segregation. The remaining flowers were allowed to mature normally and recover the R1 seed. Among 15 R1 seedlings, 9 were GUS-positive. The application of dicamba (CLARITY 200 ppm or CLARITY 3,000 ppm) further confirmed that the GUS+ phenotype and dicamba tolerance were cosegregants in R1 seedlings of GH_A24519.

[00101] Mechanized excision of the embryonic axis of cottonseed with modified steel rollers combined with mechanized recovery of explants from crushed seed, and effective selection of transformed material, including "chemical pruning", after transformation mediated by Agrobacterium in injured explants, thus allowing production of transgenic cotton plants within a short time period, 3 months, with transgenic R1 seed available within 24 weeks after transformation. The time required to obtain transgenic plants and progeny is significantly less than the time required to produce transgenic cotton plants via embryogenesis, the resulting plants are fertile, and the plants can be obtained from numerous target cotton germplasms of interest, even in strains with deficient embryogenic potential. These methods provide the advantage of increasing the efficiency and robustness of the process, while at the same time reducing costs and ergonomic burden.

Example 7 Efficiency of Manual versus Mechanized Excision.

Tabela 14. Comparação de homem-hora necessários para 10.000 ex- plantes/semana produzidos.

Tabela 15. Comparação de produtividade

[00102] The manual process of excision of the embryonic axis of the seed is slow, labor intensive and carries significant ergonomic load. The use of machine excision in combination with mechanized sieving not only overcomes the aforementioned problem, but is also amenable to scale-up to increase production volume. Table 13 and Table 14 present comparisons of manual versus progressively improved excision/mechanized sieving methods on production volume and excision productivity. Productivity for manual excision is based on the assumption of 7 hours/day, 4 days/week of uninterrupted excision, while productivity for machine excision is based on the assumption of 1 run (45-75 minutes)/day, 4 days/week. Table 15 indicates that mechanized excision/sieving can produce 4,0005,000 quality explants, i.e. explants with visible meristem, per hour, an improvement of about 20 times over manual excision and provides the advantage of higher production volume. high and lower ergonomic load.Table 13. Comparison of excision production volume.

Table 14. Comparison of man-hours needed for 10,000 explants/week produced.

Table 15. Productivity Comparison

Example 8 Effect of Explant Storage on Transformation

[00103] Storage of machine excised cotton explants was also explored. The ability to stock transformation-ready explants would allow more flexibility to program and conduct transformation experiments. After excision, the cotton ex-plants can be stored before continuing, for example, with the transformation process. In one experiment, explants were stored overnight at 4°C after excision, before inoculation with Agrobacterium. The explants were shown to remain competent for transformation after this storage scheme. Table 16 summarizes the results of another experiment. After the explants were recovered, as described, for example, in Examples 1 and 2 above, they were placed on a sterile Plantcon® (e.g., ICN Biomedical Catalog No. 26-720-02) or on filter paper moistened with medium. INO and placed in a sterile 150 mm Petri dish in a refrigerator at 4°C in the dark for the designated storage time of 0 versus 7 days. The explants were brought to room temperature before inoculation. The % Regenerable represents the percentage of the total number of explants that were able to develop into a seedling. The % GUS-positive Regenerables refers to the number of GUS-positive explants divided by the total number of regenerable explants x 100. The Regenerable % hits with strong near-periclinal GUS expression is the number of explants with strong GUS expression near periclinal divided by the total number of regenerable explants x 100.

[00104] Altogether, the results (Table 16) indicate that storing explants at 4°C for 3 days before inoculation improved the percentage recovery of regenerable explants, and therefore is a simple and effective method to control transformation workflow. Even though cold storage seemed to reduce the percentage of seedlings that expressed GUS in relation to the number of regenerable explants, those that continued to grow more likely had stable GUS expression. Consequently, the labor involved in producing transformation episodes was also reduced.Table 16. Effect of explant storage on transformation.

Example 9 Effect of Manipulating Other Parameters During Inhibition, Coculture, Selection and Growth Stages.

[00105] Manipulating one or more parameters between parameters such as reducing the imbibition temperature from 28°C to 24°C or 15°C, initial selection of explants at 35°C for 3 days after co-culture, co-culture under lighting conditions that allow normal development of plastids (e.g. an intensity > 5 μ E, e.g. about 5 μ E up to about 130-200 μ E, under a photoperiod of 16 hours of light/ 8 hours of darkness), use of higher concentration of spectinomycin during inoculation/coculture or selection, use of spectinomycin in the coculture medium, and select plants by spraying afterwards (e.g. "chemical pruning" during rooting or later growth), it was possible to increase the % of germline positive episodes per explant by 2 to 10 times. The use of spectinomycin in the pre-culture medium may also prove beneficial to increase TF (%) or to improve the quality of episodes.

Método A: Todos explantes em cada tratamento foram co ocados em um PLANTCON e o inóculo de Agrobacterium foi adicionado pra cobrir os explantes. Os explantes no inóculo passaram por ultrassom (ultras-som a granel) por 2 min e em seguida 10 min em vascolejador (80rpm). Depois, o inóculo foi removido e os explantes foram distribuídos para PLANTCON, cada um contendo um pedaço de pepel de filtro e 5 mL de meio de inoculação.[00106] In further studies, seed imbibition, and explant excision were performed as noted above (eg example 5) or as in US Patent Publication No. 2005/0005321. For inoculation and co-culture, an Agrobacterium ABI strain (C58 derivative) carrying a binary vector carrying 1 or 2 T-DNAs and comprising an aadA selectable marker gene and a uidA screenable marker was used. The explants were passed in bulk ultrasound, or ultrasound in individual PLANTCON containers. Shoot induction and selection was performed by superficially plating the explants on solid selection medium (Table 9) for about 4 weeks. A further induction of shoots and rooting was performed by growing the explants with green shoots in Oasis® buffers (Smithers-Oasis USA; Kent, OH) for shoot elongation and rooting induction from original radicals in a simple liquid medium, without selection, containing 0.5 g/L of WPM salts with vitamins ((Phytotech L449) and 0.25 mg/L of IBA for about 2-3 weeks in an oven. The results are shown in Table 17. An initial temperature of the culture of 35°C has been shown to be beneficial for the production of GUS-positive cotton buds.Table 17. Results of experiments comparing two inoculation/coculture methods, and two different culture schemes.

Method A: All explants in each treatment were placed in a PLANTCON and Agrobacterium inoculum was added to cover the explants. The explants in the inoculum underwent ultrasound (bulk ultrasound) for 2 min and then 10 min in a vascolejador (80rpm). Afterwards, the inoculum was removed and the explants were distributed to PLANTCON, each containing a piece of filter paper and 5 mL of inoculation medium.

[00107] Method B: The explants were distributed to the lid part of each PLANTCON (approximately 100 explants per PLANTCON). Five milliliters of Agrobacterium inoculum was added. The explants were then sonicated for 20 s, and were immediately transferred along with the inoculum to the bottom part of the PLANTCON, which holds a piece of filter paper.

Example 10 Repeated Transformation of Cotton Germplasm with Round-up Ready®.

[00108] Using the methods described in this Patent, superior transgenic germplasm with Round-Up Ready® can be further transformed using a vector of 2 T-DNA's (or alternatively two vectors with 1 T-DNA) that encodes the aadA gene for selection with spectinomycin and employing a new gene (often referred to as "the gene of interest"; "GOI") on the second T-DNA to allow segregation away from the aadA as described above. The following cotton method and database (dataset) are useful in describing this "repeated transformation".

[00109] The cottonseed variety RRFlex® (07W610F) and the non-transgenic germplasm control variety (00S04) were compared. The seed was soaked for ~18 h at 24°C, machine excised and machine sieved (in two steps), then enriched by explant flotation. The explants were inoculated with Agrobacterium suspension in INO at an OD 0.3, ultrasound for 2 min, and incubated for 10 min. The Agrobacterium suspension was then removed and the explants were distributed into coculture vessels at approximately 2 g per vessel. The explants were placed on filter papers moistened with 5 mL of coculture medium (INO with additions of 50 ppm nystatin, 10 ppm TBZ (thiabendazole), and 100 ppm spectinomycin) and co-cultured in a Percival incubator. illuminated at approximately 23 to 25°C (16 h light/8 h dark, light intensity > 5 μ E, such as 5 μ E at 200 μ E) for 3 days. The explants were then transferred onto selection medium (Table 9), incubated for 3 days at 35°C in a lighted room (16 h light/8 h dark), and then taken to a room lit at 28° C (16 h light/8 h dark). Phenotype-positive green seedlings were harvested 6 weeks after inoculation, placed in OASIS buffers moistened with 0.5 g/L of WPM salts and brought to greenhouse conditions. After the plants had acclimated and started to grow, they were tested for CP4, GUS, vascular expression of GUS (a predictor of germline transformation). Repeatedly transformed transgenic plants were expected to be CP4+ GUS+, while transformed control plants were expected to be CP4-GUS+. A yield analysis of transformed plants is listed in Table 18 below. The described procedure can thus be used for repeated transformation of transgenic cotton plants with an efficiency similar to the transformation of a conventional non-transgenic cotton variety.Table 18. Frequency of Transformation Observed from Repeated Transformation of Germplasm of Transgenic Cotton.

[00110] 1 The compositions and honey all described and claimed herein can be made and performed without undue experimentation in light of the present specification. While the compositions and methods of this invention have been described in terms of the foregoing illustrative embodiments, it should be apparent to those skilled in the art that variations, changes, modifications and alterations can be applied to the composition, methods, and steps or in the sequence of steps of the methods described herein, without departing from the true concept, spirit and scope of the invention. More specifically, it should be apparent that certain agents that are chemically and also physiologically related can replace the agents described herein, and at the same time, the same or similar results could be achieved. All such substitutes and modifications evident to those skilled in these techniques are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims (38)

1. A high-yield method for producing transformed cotton plant tissue, characterized in that it comprises: (a) mechanically breaking cotton seeds to obtain a plurality of embryonic cotton meristem explants; and (b) contacting the explants with a selected DNA sequence encoding a selectable marker to obtain at least a first explant transformed with the selected DNA; (c) regenerating from at least the first explant transformed with the selected DNA a chimeric cotton plant comprising transgenic plant tissue; (d) selecting transgenic tissue from the chimeric cotton plant by contacting the chimeric cotton plant with a selective agent, wherein the selectable marker confers tolerance to the selective agent; and (e) obtaining a non-chimeric transgenic cotton plant from the transgenic tissue selected in step (d). 2. Method according to claim 1, characterized in that the explants are stored at a temperature between 0-15°C for between 1 hour and 7 days before step (b). 3. Method according to claim 1, characterized in that the resulting plant is chimeric. 4. Method according to claim 1, characterized in that it further comprises screening the explants before placing the explants in contact with the selected DNA sequence to identify a fraction of explants susceptible to transformation with the selected DNA and regeneration of a transgenic plant therefrom, wherein screening the explants comprises: (i) placing the mechanically ruptured cotton seeds comprising the explants in an aqueous environment and selecting the explants to place in contact with the selected DNA, based on flotation; or (ii) sieving the mechanically ruptured cottonseeds to remove explants from the seed coat or cotyledon tissue. 5. Method according to claim 4, characterized in that the explant screening comprises enriching the fraction of explants susceptible to transformation. 6. Method according to claim 1, characterized in that the explants of step (a) comprise a transgene. 7. Method according to claim 1, characterized in that it further comprises regenerating chimeric transgenic plant tissue from the explant and selecting or screening the transgenic tissue from the plant tissue. 8. Method according to claim 7, characterized in that it further comprises regenerating a chimeric plant from the explant and selecting or screening transgenic tissues from the plant. 9. Method according to claim 7, characterized in that the selection or screening of transgenic tissue comprises contacting the tissue with a selective agent or an agent that produces a selectable phenotype, selected from the group consisting of glufosinate, dicamba, glyphosate, spectinomycin, streptomycin, kanamycin, G418, paromomycin, imidazolinone, a substrate of GUS, and combinations thereof. 10. Method, according to claim 1, characterized in that the mechanical disruption of cotton seeds comprises passing the seeds through rollers that crush the seeds. 11. Method according to claim 10, characterized in that the rollers comprise secondary grooves. 12. Method according to claim 10, characterized in that the rollers are made of stainless steel. 13. Method according to claim 1, characterized in that placing the explants in contact with a selected DNA sequence comprises placing the explants in contact with Rhizobium or Agrobacterium spp. recombinants capable of transforming at least one first cell of the explant with the selected DNA. 14. Method according to claim 13, characterized in that it comprises placing the explant in contact with a recombinant Agrobacterium culture developed to an OD660 between 0.0045 and 1.4. 15. Method according to claim 13, characterized in that it comprises placing the explant in contact with an Agrobacterium culture, in which the pH at which the meristematic cotton tissue is placed in contact by the Agrobacterium cells is between 5, 0 and 10.0. 16. Method according to claim 1, characterized in that putting the explants in contact with a selected DNA sequence comprises transforming the explants into microprojectile bombardment. 17. A method for producing trans-formed cotton plant tissue, comprising: (a) contacting a plurality of cotton embryonic meristem explants with a Rhizobium or Agrobacterium spp. recombinant capable of transforming at least a first cell of the explant with a selected DNA sequence encoding a selectable marker providing tolerance to a selective agent; (b) transferring the plurality of explants to a first culture medium comprising the selective agent; and then (c) visually selecting at least one first explant transformed with the selected DNA based on a screenable phenotype conferred by the selectable marker in the presence of the selective agent; and (d) transferring at least the first visually selected explant to a second medium. 18. Method according to claim 17, characterized in that the selective agent is an antibiotic. 19. Method according to claim 17, characterized in that the selectable marker comprises an adenylyl transferase. 20. Method according to claim 19, characterized in that the selected DNA sequence comprises aa- dA. 21. Method according to claim 18 or 20, characterized in that the selective agent is spectinomycin. 22. Method according to claim 17, characterized in that the second means is a means of regeneration. 23. Method according to claim 22, characterized in that the second means is a rooting means. 24. Method according to claim 17, characterized in that it further comprises stimulating the explant to form multiple shoots during, or before, the transfer of explants to the first culture medium. 25. A high-yield method for producing transformed cotton plant tissue, characterized in that it comprises: (a) contacting a plurality of cotton embryonic explants with a Rhizobium or Agrobacterium spp. recombinant capable of transforming at least a first cell of the explant with a selected DNA sequence encoding a selectable marker to obtain at least a first explant transformed with the selected DNA; (b) regenerating a chimeric cotton plant from at least the first explant transformed with the selected DNA while transgenic tissue from the chimeric cotton plant is selected by contacting the chimeric cotton plants with a selective agent, wherein the selectable marker confers tolerance to the selective agent; and (c) obtaining a non-chimeric transgenic cotton plant from the transgenic tissue selected in step (b). 26. Method according to claim 1 or 25, characterized by the fact that the regeneration of the transgenic cotton plant does not comprise the generation of a callus culture from the explant. 27. A high-yield method for producing transformed cotton plant tissue, characterized in that it comprises: (a) contacting a plurality of cotton embryonic explants with a Rhizobium or Agrobacterium spp. recombinant capable of transforming at least a first cell of the explant with a selected DNA sequence encoding a selectable marker to obtain at least a first explant transformed with the selected DNA; (b) selecting the transgenic tissue by contacting the transformed explant with a selective agent, wherein the selectable marker confers tolerance to the selective agent; (c) regenerating from at least the first explant transformed with the selected DNA a chimeric cotton plant comprising transgenic plant tissue; and (d) obtaining a non-chimeric transgenic cotton plant from the transgenic tissue regenerated in step (c); in which the regeneration of the transgenic cotton plant does not comprise the generation of a callus culture from the explant. 28. Method according to claim 25 or 27, characterized in that the explants are stored at a temperature between 0-15 °C for between 1 hour and 7 days before step (a). 29. Method according to claim 25 or 27, characterized by the fact that the transgenic cotton plant arises from the transformation of a meristem that results in the transformation of germinal tissue. 30. Method according to claim 25 or 27, characterized in that the selected DNA sequence further encodes a screenable marker, or specifies an agronomic trait. 31. Method according to claim 25 or 27, characterized in that the explants comprise a transgene before step (a). 32. Method according to claim 7, 25 or 27, characterized by the fact that the transgenic tissue of the cotton plant arises from the transformation of the meristem. 33. Method according to claim 13, 25 or 27, characterized in that Rhizobium or Agrobacterium spp. are suspended in the presence of a selective agent active against an untransformed explant before contacting the explants with a recombinant Rhizobium or Agrobacterium spp. 34. Method according to claim 13, 25 or 27, characterized in that, after contacting the explants with a selected DNA sequence, the explants are cultured in the presence of a selective agent at 35 °C or the explants are cultured. under lighting conditions that allow normal plastid development. 35. Method according to claim 24, characterized in that the growth at 35 °C is carried out for 1-7 days; the selective agent is selected from the group consisting of spectinomycin, streptomycin, kanamycin, glyphosate, glufosinate, hygromycin and dicamba; or explants are cultured with a 16-hour light/8 dark photoperiod. 36. Method according to claim 13, 25 or 27, characterized in that the explants are cultivated in the presence of a fungicide before, during or after step (a). 37. Method according to claim 36, characterized in that the explants are grown in the presence of a fungicide and DMSO. 38. Method according to claim 37, characterized in that the explants are cultured in the presence of nystatin, thiabendazole and DMSO.

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